全球蛋白质组分析受限于低丰度肽段或特定理化特性的鉴定。本文开发并优化了一维在线碱性pH反相纳米电喷雾串联质谱（碱性pH-MS/MS）方法，用于全局蛋白质组学分析。在该方法中，肽段在具有碱性pH流动相（pH=8.0）的纳米C18柱上分离，并直接注入质谱仪中。碱性pH-MS/MS与传统的在线低pH反相纳米电喷雾串联质谱（低pH-MS/MS）的独特肽段重叠率低至45％，强烈表明这两种方法互补。此外，碱性pH-MS/MS还显示了更高比例的具有负平均疏水性（GRAVY）或高等电点（pI）的肽段的鉴定能力。与低pH-MS/MS相比，碱性pH-MS/MS可以对组氨酸、赖氨酸、甲硫氨酸和脯氨酸肽段进行富集选择。同时，本研究进一步证明了碱性pH-MS/MS和低pH-MS/MS在15种肝内胆管癌（iCCA）细胞系的酸性胰酶消化产物分析中互补性。交替使用60分钟的碱性pH-MS/MS和60分钟的低pH-MS/MS方法在氨基酸变异和蛋白质组鉴定的精度方面优于传统的120分钟低pH-MS/MS方法。因此，本研究将碱性pH-MS/MS方法确立为全局蛋白质组学分析的简单、竞争性和替代方法。

The global proteome analysis was limited by the identification of peptides with low abundance or specific physiochemical properties. Here, a one-dimensional online alkaline-pH reverse phase nanoelectrospray-tandem mass spectrometry (alkaline-pH-MS/MS) method was developed and optimized for global proteomic analysis. In this method, peptides were separated on a nanoflow C18 column with an alkaline-pH mobile phase (pH = 8.0) and directly injected into the mass spectrometer. The unique peptides overlapped between alkaline-pH-MS/MS and conventional online low-pH reverse phase nanoelectrospray-tandem mass spectrometry (low-pH-MS/MS) were as low as 45%, strongly indicating that these two methods were complementary to each other. In addition, alkaline-pH-MS/MS showed identification capacity for a higher proportion of peptides with negative grand average of hydropathy (GRAVY) or high isoelectric point (pI). Compared to low-pH-MS/MS, alkaline-pH-MS/MS enabled enrichment preference toward histidine-, lysine-, methionine-, and proline-containing peptides. The complementarity of alkaline-pH-MS/MS and low-pH-MS/MS was further demonstrated for the analysis of tryptic digests from 15 intrahepatic cholangiocarcinoma (iCCA) cell lines. The alternating 60 min alkaline-pH-MS/MS plus 60 min low-pH-MS/MS method outperformed the conventional 120 min low-pH-MS/MS method in both the identification of amino acid variants and protein groups. Therefore, we established the alkaline-pH-MS/MS method as a simple, competitive, alternative method to low-pH-MS/MS for global proteomic analysis.